Protein liquid–liquid phase separation (LLPS), a pivotal phenomenon intricately linked to cellular processes, is regulated by various other proteins. However, there is still a lack of high-throughput methods for screening protein regulators of LLPS in target proteins. Here, we developed a CRISPR/Cas9-based screening method to identify protein phase separation regulators by integrating bimolecular fluorescence complementation (BiFC) and fluorescence-activated cell sorting (FACS). Using this newly developed method, we screened the RNA-binding proteins that regulate PABPN1 phase separation and identified the tumor suppressor QKI as a promoter of PABPN1 phase separation. Furthermore, QKI exhibits decreased expression levels and diminished nuclear localization in colorectal cancer cells, resulting in reduced PABPN1 phase separation, which, in turn, promotes alternative polyadenylation (APA), cell proliferation, and migration in colorectal cancer.

Hepatocellular carcinoma (HCC) ranks among the top causes of cancer-related mortality worldwide, and its complex tumor microenvironment (TME) contributes to poor responses to immunotherapy. Although PD-1/PD-L1 blockade has emerged as an effective treatment strategy, therapeutic resistance frequently limits its clinical benefit. Here, we uncover a distinct macrophage population associated with anti-PD-1 resistance in HCC. Single-cell transcriptomic profiling reveals an NFKBIZ⁺ M0 macrophage subset predominantly present in non-responders. Notably, these macrophages exhibit a hypoxia-induced phenotype characterized by the secretion of VEGFA and HBEGF, which cooperatively enhance tumor angiogenesis, alongside an elevated expression of the inflammatory chemokines CXCL2, CXCL3, and CXCL8 that consolidate an immunosuppressive, pro-tumorigenic microenvironment. Transcriptional regulatory network analysis further identified FOSB–VEGFA and FOS–HBEGF axes as key drivers of this pathogenic macrophage phenotype. Our findings define a distinct NFKBIZ⁺ macrophage population that mechanistically links hypoxia, angiogenesis, and immune evasion to PD-1 blockade resistance. This work provides new insights into the cellular and molecular basis of immunotherapy failure in HCC and highlights potential targets for overcoming treatment resistance.